大鼠CD47 RNAi腺病毒载体的构建与鉴定

发布时间：2018-07-28 14:35

【摘要】：目的:构建并鉴定大鼠CD47 RNAi腺病毒载体,制备具有一定滴度含CD47 RNAi基因的重组腺病毒,为后期研究CD47 RNAi在慢性心力衰竭中的作用及其分子机制奠定基础。方法:根据大鼠CD47基因序列,设计并合成3对CD47基因的特异性小干扰RNA(siRNA),合成互补的寡核苷酸链,构建大鼠CD47 RNAi腺病毒载体,并对目的基因进行鉴定。通过脂质体2000介导CD47 RNAi腺病毒载体和包装质粒共转染人HEK293细胞,以得到重组腺病毒,对其进行扩增、纯化及滴度测定。结果:成功构建了大鼠CD47 RNAi腺病毒载体。经重组腺病毒扩增与纯化,测得最终获得的重组腺病毒滴度为1×10~(11) PFU/ml。结论:成功构建获得了具有一定滴度含CD47 RNAi基因的重组腺病毒,为后期顺利开展有关CD47 RNAi在大鼠慢性心力衰竭中的作用及分子机制研究提供了研究基础。
[Abstract]:Aim: to construct and identify the adenovirus vector of rat CD47 RNAi and to prepare recombinant adenovirus containing CD47 RNAi gene titer in order to study the role of CD47 RNAi in chronic heart failure (CHF) and its molecular mechanism. Methods: according to the sequence of rat CD47 gene, three pairs of CD47 gene specific small interfering RNA (siRNA), were designed and synthesized to synthesize complementary oligonucleotide chains. The adenovirus vector of rat CD47 RNAi was constructed, and the target gene was identified. CD47 RNAi adenovirus vector and packaging plasmid were co-transfected into human HEK293 cells by liposome 2000 to obtain recombinant adenovirus. The recombinant adenovirus was amplified, purified and titer determined. Results: the adenovirus vector of rat CD47 RNAi was successfully constructed. After amplification and purification of recombinant adenovirus, the titer of recombinant adenovirus was 1 脳 10 ~ (11) PFU / ml. Conclusion: the recombinant adenovirus with certain titer of CD47 RNAi gene was successfully constructed, which provides a basis for the study of the role and molecular mechanism of CD47 RNAi in chronic heart failure in rats.
【作者单位】：三峡大学第一临床医学院心内科三峡大学心血管病研究所;
【基金】：国家自然科学基金(No:81470387)
【分类号】：R541.6

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